Cell morphology and CFU-F frequency of cultured hES-MP002.5 cells compared with BM-MSCs.

<p>hES-MP002.5 cells and BM-MSCs, respectively, were assayed for CFU-F. (A–B) Except for differences in cell size, colonies derived from hES-MP002.5 cells (A) showed a similar morphology compared to CFU-F assayed from standard bone marrow-derived MSCs (B). Scale bars indicate 100 μm. (C) Frequencies of CFU-F in standard BM-MSCs preparations are higher when compared with hES-MP002.5 cells. Results are shown as mean number of CFU-F numbers per 100 seeded cells ± standard deviation. Data were analyzed by student's <i>t test</i>. n = 6. * p<0.05.</p

Differentiation capacity of hES-MP002.5 cells and BM-MSCs.

<p>Cultured cells were differentiated towards the adipogenic (left column), osteogenic (middle column) and chrondrogenic lineage (right column). Formation of adipocytes was confirmed by Oil-Red-O staining of lipid vacuoles in differentiated BM-MSCs (A) and in hES-MP002.5 cells (B). Undifferentiated hES-MP002.5 cells controls are shown in (C). Osteoblastic differentiation was demonstrated by calcium deposits stained by von Kossa staining of differentiated BM-MSCs (D) and hES-MP002.5 cells (E), but not in the undifferentiated hES-MP002.5 cells control (F). Differentiation into chondrocytes was confirmed by staining for aggrecan (red) in sectioned chondrocyte pellets derived from differentiated BM-MSCs (G) and hES-MP002.5 cells (H). Undifferentiated hES-MP002.5 cells are shown as control in (I). Black scale bars indicate 50 μm; white scale bars indicate 500 μm.</p

Immune-modulatory functions of BM-MSCs and hES-MP002.5 cells.

<p>BM-MSCs or hES-MP002.5 cells were added to mixed lymphocyte cultures (A) and PHA-stimulated lymphocyte cultures (B) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055319#pone.0055319-Ahrens1" target="_blank">[22]</a> to examine their effect on allogeneic- and mitogen-induced lymphocyte proliferation, respectively. The percentage of stromal cells added to the cultures ranged from 0% (control) to 20% (x-axis). Lymphocyte proliferation rates are expressed as percentages of controls, i.e. cultures to which no MSCs were added. BM-MSCs for these experiments were generated as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055319#pone.0055319-LeBlanc3" target="_blank">[26]</a>. Data are shown as mean ± SD of at least 3 independent experiments. Statistical differences compared to controls are indicated as *: p<0.05 (student <i>t test</i>).</p

Surface marker expression profiles of hES-MP002.5 cells and BM-MSCs.

<p>hES-MP002.5 cells and BM-MSCs were trypsinized and stained with different combinations of antibodies for flow cytometric analysis. The data are presented as histograms. The percentage of the maximum of the number of cells in each channel is shown on the y-axis. Samples are presented in the shaded plots, corresponding isotype controls are shown as open lines. hES-MP002.5 cells and BM-MSCs showed comparable surface marker expression profiles, except for differences in CD90 expression. Representative histograms of one of three independent experiments are shown.</p

Analysis of in-vivo stromal function of BM-MSCs and hES-MP002.5 cells by co-transplantation with CD34+ hematopoietic cells in NSG mice.

<p>NSG mice received intrafemoral transplantation of stromal cells into the left femur followed by intravenous injections of cord blood derived CD34+ cells. After 8 weeks, cells from both femurs were harvested separately and stained with anti human CD45 (A, C) and CD34 (B, D) antibody and analyzed by flow cytometry. Control mice received intrafemoral PBS only. Data are presented as percentages (A, B) as well as absolute numbers (C, D) of human cells in the femurs of individual mice. Lines connect data from left (injected, BM-MSC+, hES-MP002.5 cells+, PBS+, shown with solid dots) and right (non-injected, BM-MSC-, hES-MP002.5 cells-, PBS-, shown with circles) femurs of the same animal. Mean values were indicated as horizontal lines. Statistical differences are indicated as: * <i>p</i><0.05.</p

Acoustic Enrichment of Extracellular Vesicles from Biological Fluids

Extracellular vesicles (EVs) have emerged as a rich source of biomarkers providing diagnostic and prognostic information in diseases such as cancer. Large-scale investigations into the contents of EVs in clinical cohorts are warranted, but a major obstacle is the lack of a rapid, reproducible, efficient, and low-cost methodology to enrich EVs. Here, we demonstrate the applicability of an automated acoustic-based technique to enrich EVs, termed <i>acoustic trapping</i>. Using this technology, we have successfully enriched EVs from cell culture conditioned media and urine and blood plasma from healthy volunteers. The acoustically trapped samples contained EVs ranging from exosomes to microvesicles in size and contained detectable levels of intravesicular microRNAs. Importantly, this method showed high reproducibility and yielded sufficient quantities of vesicles for downstream analysis. The enrichment could be obtained from a sample volume of 300 μL or less, an equivalent to 30 min of enrichment time, depending on the sensitivity of downstream analysis. Taken together, acoustic trapping provides a rapid, automated, low-volume compatible, and robust method to enrich EVs from biofluids. Thus, it may serve as a novel tool for EV enrichment from large number of samples in a clinical setting with minimum sample preparation